The cytotoxic effect of 5-azacytidine (AzaCR) on normal hematopoietic colony-forming units (NCFU) and L1210 leukemic colony-forming units (LCFU) in the femoral marrow of BALB/c x DBA/2 F1 mice was studied using the spleen colony assay. Dose-survival curves for LCFU and NCFU were biphasic. Repopulation of LCFU was rapid at a low dose of AzaCR (0.1 mg/mouse) but was delayed for greater than 6 days at higher doses (0.25 mg/mouse and above). Of the agents tested in this system, only AzaCR exhibited these properties. Survival of mice with L1210 leukemia following AzaCR administration was prolonged beyond that predicted by the degree of LCFU reduction alone, and reflected the delay in LCFU repopulation. In contrast, repopulation of NCFU in normal mice was not delayed at a high dose of AzaCR (0.5 mg/mouse). AzaCR produced a nine-fold greater reduction of NCFU in leukemic mice than in normal mice, measured 5 days after AzaCR injection. While divided doses of AzaCR produced LCFU cytotoxicity equivalent to a single dose, 24-hr infusions of high doses were inferior to single infections.